Previous reports from our laboratory have found that nerve growth factor differentiated pheochromacytoma cell lines (NGFDPC12 cells) exposed to pathological levels of exogenous palmitic (PA) and stearic (SA) fatty acids -- at a level comparable to that found during hypoxia/ischemia - causes a dramatic decrease in cell viability and apoptosis. This decrease in cell survival occurs because these saturated FFA induced massive apoptosis (program cell death) during 24 hours exposure. In contrast, similar levels of arachidonic (AA) or oleic (OL), EPA or DMA FFA did not decrease the viability of these cells. The central hypothesis states that high levels of PA or SA fatty acids induce neuronal apoptosis secondary to mitochondria! dysfunction. This application will primarily focus on characterizing the apoptotic effect of PA and SA in NGFDPC12 cells and extend this analysis to primary retinal ganglion and cortical cell neurons. Specific Aim 1 will include the characterization of PA and SA-mediated caspase-independent neuronal cell death. This aim will determine the effects of these saturated FFA in the survival of rat cortical and retinal ganglion primary cells in culture. The experimental design will evaluate whether the loss of cell viability is result of apoptosis or necrosis and will determine whether the process is dependent on caspase activation. Further experiments will analyze what specific apoptotic proteins are involved in the process. Specific Aim 2 will determine whether FFAmediated neuronal cell death occurs through the mitochondrial pathway. Preliminary cDNA array analysis and Quantitative real time PCR data has identified several candidate mitochondrial genes that are involved in this process. This aim will elucidate the role of these genes by using siRNA knockdown and overexpression to modulate cellular levels. We anticipate that these studies will help us to clarify the mechanisms of how pathological levels of saturated FFA affect neuronal survival and apoptosis.